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COD 22047

2 x 60 mL + 2 x 12 mL

HEMOGLOBIN A1C-DIRECT
(HbA1C-DIR)

COD 22147
1 x 60 mL + 1 x 12 mL

STORE AT 2-8C
Reagents for measurement of Hemoglobin A1C concentration
Only for in vitro use in the clinical laboratory

HEMOGLOBIN A1C-DIRECT (HbA1C-DIR)


DIRECT

PRINCIPLE OF THE METHOD


After preparing the hemolysate, the Hemoglobin A1C (HbA1C) concentration is quantified by a
latex turbidimetric assay. The different hemoglobins present in the hemolysate are unspecifically
adsorbed on the latex particles surface in a ratio equivalent to their concentration in the sample.
The addition of an anti-human HbA1C antibody causes agglutination that is proportional to the
concentration of hemoglobin A1C and can be measured by turbidimetry.

CONTENS
A. Reagent
B. Reagent

COD 22047

COD 22147

2 x 60 mL
2 x 12 mL

1 x 60 mL
1 x 12 mL

COMPOSITION
A. Reagent. Suspension of latex particles, sodium azide 0.95 g/L, pH 8.0.
B. Reagent. Anti-human HbA1C antibody, stabilizers, pH 6.0.

STORAGE
Store at 2-8C.
Reagents are stable until the expiry date shown on the label when stored tightly closed and if
contaminations are prevented during their use.
Indications of deterioration: absorbance of the blank over 0.500 at 670 nm.

AUXILIARY REAGENTS

REAGENT PREPARATION
Reagents are provided ready to use.
Opened reagents and stored in the analyzer chamber at 2-8C are stable 30 days.

SAMPLES
Venous blood collected by standard procedures and with EDTA as anticoagulant.
HbA1C in blood is stable 7 days at 2-8C.

REFERENCE VALUES
The following cut-off points have been established by the Diabetes Control and Complications
Trial Research Group (DCCT) and have been adopted by many countries for a reference
population (Non diabetic) and for the evaluation of the degree blood glucose control in diabetic
patients1,2.
IFCC mmol/mol)

NGSP-DCCT (%)

Reference values/Degree of control

20 - 48
42 - 53
53 - 64
> 64

4.0 - 6.5
6.0 - 7.0
7.0 - 8.0
> 8.0

Non Diabetic
Goal
Good Control
Action suggested

CALIBRATION
It is recommended to do a reagent blank every day and a calibration at least every 30 days, after
reagent lot change or as required by quality control procedures.

PROCEDURE
Hemolysate preparation
The calibrators should be treated as patient samples.
1. Pipette into a test tube:
Blood

It is recommended to use the Hemoglobin A1C Controls, Normal (cod. 18001) and Elevated (cod.
18002), to verify the performance of the measurement procedure.
Each laboratory should establish its own internal Quality Control scheme and procedures for
corrective action if controls do not recover within the acceptable tolerances.

METROLOGICAL CHARACTERISTICS
These metrological characteristics have been obtained using BA400 analyzer. Results may vary
if a different instrument or a manual procedure are used.
Detection limit: 2 mmol/mol.
Measurement interval: (approximate value dependent on the highest standard concentration):
2 - 140 mmol/mol. For higher values dilute sample 1/2 with distilled water and repeat
measurement.
Repeatibility (within run):
Mean concentration

CV

37 mmol/mol
78 mmol/mol

1.8 %
0.8 %

20
20

1000 L

2. Mix gently. Avoid the formation of foam. Incubate at room temperature for 5 minutes.
The hemolysate is stable 72 hours at 2-8C.
Assay parameters (Notes 1 and 2)
These reagents may be used in several automatic analysers. Specific instructions for application
in many of them are available on request.
Wavelength:
670 nm
Temperature:
37C
Analysis mode: 2 points

Sample / Standard
Distilled water
Reagent A

Blank

Sample / Standard

3 L
190 L

3 L
190 L

40 L

40 L

Mix and incubate 3-5 minutes. Then add:

Mix and read the absorbance after 10 seconds and after 5 minutes.

CALCULATION
The HbA1C values obtained are traceable to IFCC Reference Method.

Mean concentration

CV

37 mmol/mol
78 mmol/mol

2.9 %
1.3 %

25
25

Trueness: Results obtained with this reagent did not show systematic differences when
compared with reference reagents. Details of the comparison experiments are available on
request.
Interferences: Lipemia (triglycerides 4g/L) and bilirubin (10 mg/dL) do not interfere. Other
drugs and substances may interfere4.
In the immunoassay methods, the presence of acetylated-Hb, carbamylated-Hb, labile HbA1C
HbE and HbD do not affect the results5,6. Other hemoglobin variants like HbS, HbF or HbC
can interfere 5.
In hemolytic anemia, iron deficiency anemia and transfusion, the average age of erythrocytes
is altered. Caution should be used when interpreting the HbA1C results from patients with
these conditions.

DIAGNOSTIC CHARACTERISTICS
HbA1C is the product of the irreversible condensation of glucose with the N-terminal residue of
the -chain of hemoglobin A.
The HbA1C concentration in blood is directly proportional to the mean concentration of glucose
prevailing in the previous 6-8 weeks, equivalent to the lifetime of the erythrocytes1, and the
estimated average glucose (eAG) during this period can be calculated with the formulas below7.
eAG (mg/dL) = 28.7 x HbA1C-NGSP-DCCT (%) 46.7
eAG (mmol/L) = 1.59 x HbA1C-NGSP-DCCT (%) 2.59
eAG (mg/dL) = 2.64 x HbA1C-IFCC (mmol/mol) + 15.0
eAG (mmol/L) = 0.146 x HbA1C-IFCC (mmol/mol) + 0.843
HbA1C levels are a valuable adjunct to glucose determinations in the assessment and follow up
of individuals with diabetes mellitus, providing much more reliable information for glycaemia
monitoring than do determinations of glucose. Numerous studies have shown that diabetes
related complications may be reduced by the long term monitoring and tight control of blood
glucose levels. The HbA1C concentration may also be a useful tool in the diagnosis of
diabetes8.
Clinical diagnosis should not be made on the findings of a single test result, but should integrate
both clinical and laboratory data.

NOTES

10 L

Distilled water

M22047i-02

QUALITY CONTROL

Reproducibility (run to run):

S. HbA1C Direct Standards (Cod. 31048). 4 levels for 0.5 mL. Human blood. HbA1C
concentration is given on the label.
Human blood used in the preparation of the standard has been tested and found to be
negative for the presence of antibodies anti-HIV and anti-HCV, as well as for HBs antigen.
However, the standard should be handled cautiously as potentially infectious.
Reconstitute with 0.5 mL of distilled water. Stable for 30 days at 2-8C.

Reagent B

The traceable values to Reference Method as described by the US National Glycohemoglobin


Standardization Program (NGSP) are calculated using the following general formula3.
%HbA1C-NGSP-DCCT (%) = 0.0915 x HbA1C-IFCC (mmol/mol) + 2.15

1. To avoid possible interferences by other assays performing HbA1C determinations in


different runs of the other test is recommended.
2. To avoid possible interferences by the analyser system liquid, it is recommended to use
distilled water as system liquid.

BIBLIOGRAPHY
1. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th ed. Burtis CA, Ashwood ER, Bruns DE.
WB Saunders Co, 2005.
2. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes
on the development and progression of long term complications in insulin-dependent diabetes mellitus. N
Engl J Med 1993; 329: 977-986.
3. Hoezel W et al. IFCC reference systems for measurement of hemoglobin A1C in human blood and the
national standarization schemes in the United States, Japan and Sweden: a method-comparison study. Clin
Chem 2004;50:166-174.
4. Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC Press, 2000.
5. Bry L, Chen P, Sacks D. Effects of hemoglobin variants and chemically modified derivates on assays for
glycohemoglobin. Clin Chem 2001;41:153-163.
6. Little R.R., Rohlfing C; Hanson S, Connolly S, Higgings T, Weykamp C, DCosta M, Luzzi V, Owen W,
Roberts WL. Effects of Hemoglobin (Hb) E and HbD traits on measurements of Glycated Hb (HbA1C) by 23
methods. Clin Chem 2008, 54: 1277-1282.
7. Nathan DM, et al. Translating the A1C assay into estimated average glucose values. Diabetes Care 2008;
31: 1473-1478.
8. Nathan DM, et al. International Expert Committee report on the role of the HbA1C assay in the diagnosis of
diabetes. Diabetes care 2009: 32: 1327-1334.

BioSystems S.A. Costa Brava, 30. 08030 Barcelona (Spain)


Quality System certified according to
EN ISO 13485 and EN ISO 9001 standards

03/2015